Placental abnormalities have been propose as a major cause for developmental defects of animals clone from differentiated somatic cells. In mice and cattle, X chromosome inactivation (XCI) is established at blastocyst stage with the paternal X chromosome (XP) preferentially inactivated (XiP) in the trophectoderm. Abnormal XCI has been found to have deleterious effect on placental growth and causes fetal death. The abnormal XCI may be caused by the impaired ability of cloned embryos to erase epigenetic marks of XCI in the donor cells, which interfere with the re-establishment of new imprinting marks. Here the investigators propose to test the hypothesis that the avoidance of both the reversal of epigenetic marks of XCI during nuclear reprogramming as well as the conflict between epigenetic marks for inactivation (Xi) and maternal origin (Xm) should improve the reprogramming of the donor nuclei and result in normal expression of X-linked genes in cloned embryos. Conversely, embryos in which Xi becomes Xa or where Xim persists in the trophectoderm will have more abnormal gene expression and poorer embryo development. Therefore, embryos in which the epigenetic marks in trophectoderm are reprogrammed to those mimicking the placenta in natural reproduction, i.e., XiP, should have the best embryo development and normal expression of X-linked genes. The Specific Aims of this application are 1, to compare blastocyst development of cloned embryos which "maintained" versus reversed the XCI patterns of the somatic donor cells. Fibroblast cells from a female donor cow with known XCI patterns will be individually cultured, clonally expanded and categorized into two groups: XipXam or XaPXim. Nuclear transfer will be conducted using these two groups of cells and the blastocyst development of the resulting embryos in which reversal and "maintenance" of the XCI pattern in the donor cells will be compared. 2, to compare the patterns and levels of expression of l0 X-linked genes in the two groups of embryos generated from Specific Aim 1. These genes include MAOA, XIST. ELF4, FACL4; PLP1, PPEF1, SYP, SYNl, BGN, and TBLl. In vivo produced XX embryos will be used as controls for normal gene expression. Aberrant expression of all of these genes (except for TBLl which escapes XCI) was found in the investigators' died bovine clones. Findings from this pilot project should generate sufficient initial data on the mechanisms of epigenetic reprogramming in cloned embryos during early embryogenesis which could lead to a full-blown investigation on the reprogramming of X-linked genes and other epigenetic marks during peri- and post-implantation development.